Infection by hepatitis viruses such as hepatitis B virus (HBV) and hepatitis C virus (HCV) is a worldwide health problem.
The World Health Organization (WHO) lists hepatitis B as the ninth leading cause of death worldwide and it has been estimated that 300 million people are chronic carriers of the virus (Hoffnagle N. Engl. J. Med. 1990, 323:337-339). Estimates of hepatitis B infection in the United States alone reaches one million. Chronic hepatitis B infection leads to cirrhosis and hepatocellular carcinoma and, if left untreated, death.
The HBV virion comprises an envelope and a nucleocapsid containing a circular, partially double stranded DNA which replicates via an RNA intermediate (Tiollais Nature [London] 1985, 317:489-495). The envelope carries the hepatitis B surface antigen, and the nucleocapsid is formed by the hepatitis B core antigen. When virions are present in the blood, an additional antigen, hepatitis B e antigen is detected. Studies have suggested that HBV is not directly cytopathic and that the host immune response to viral antigens presented on the plasma membrane of infected liver cells may play an important role in the pathogenesis of the virus (Mondelli et al. J. Immunol. 1982, 129:2773-2778; Chisari et al. Microb. Pathol. 1989, 6:311-325). Despite the progress in understanding the course of the infection, the molecular mechanisms responsible for hepatocyte death and viral clearance are not well understood because of the narrow host range of HBV and its nontransmissibility in routine cell culture systems.
The chronic carrier state has been successfully established by experimental infection of chimpanzees with HBV or HBV DNA. However, infection in these animals is self-limiting. In addition, chimpanzees are both expensive and endangered (Barker et al. J. Infect. Dis. 1973, 127:648-662; Will et al. Proc. Acad. Natl. Sci. 1985, 82:891-895).
HBV-like viruses have also been isolated and characterized in other animals such as ground squirrels, wood chucks, ducks, and tree squirrels (Feitelson et al. Proc. Natl. Acad. Sci. 1986, 83:2233-2237; Marion et al. Proc. Natl. Acad. Sci. 1980, 77:2941-2945; Mason et al. J. Virol. 1980, 36:829-836; Summers et al. Proc. Natl. Acad. Sci. 1978, 75:4533-4537). These model systems have provided much information about the biology of the HBV, including information regarding the carrier state. However, the expense and difficulty in handling these animals has limited their use.
More recently, attempts have been made to develop a model system using transgenic mice. However, in all of these transgenic lines, the mice are tolerant to HBV and disease does not develop (Takashima et al. Immunology 1990, 75:398-405; Moriyama et al. Science 1990, 248:361-364; Farza et al. J. Virol. 1988, 62:4144-4152; Araki et al. Proc. Natl. Acad. Sci. 1989, 86:207-211). Further, the induction of hepatitis in these mice has required multi-step procedures, such as the priming of lymphocytes with HBV protein in syngeneic mice and the adoptive transfer of these cells in vivo (Moriyama et al. Science 1990, 248:361-364). This tolerance to HBV makes it difficult to explore the relationship between immune responses and the overall host-virus relationship in these models. Hence, the host-virus relationship in these mice does not resemble that of human carriers with chronic liver disease. In addition, these transgenic lines are limited as model systems because they replicate virus at low levels (Farza et al. J. Virol. 1988, 62:4144-4152; Araki et al. Proc. Natl. Acad. Sci. 1989, 86:207-211). Further, the types of viral antigens present and replication of virus in the transgenic animals do not parallel that of a "natural infection". Araki et al. and Farza et al. indicate that this is due to a large region of the HBV DNA in the non-expressing transgenic animals being methylated, i.e., not capable of being expressed. Araki et al. also suggest that integration sites of the introduced DNA on the host chromosome may also have affected expression, resulting in a non-typical HBV infection. Modification of the injected sequence to improve virus expression (to parallel a normal infection) in transgenic animals is suggested.
Transgenic mice that demonstrate a high level of HBV replication comparable to that of infected livers of patients with chronic hepatitis were developed by Guidotti et al. (J. Virol. 1995, 69:6158-6169) . Guidotti et al. showed expression of a specific transcript which is not indicative of normal HBV infection. These transgenic mice also fail to show progression to a disease state, again limiting their usefulness as a model system.
Another approach to generating a hepatitis animal model has been directly injecting HBV DNA into the liver of animals and monitoring the progression of the infection. For example, rat livers were transfected in vivo with a replication competent HBV construct using a cationic lipid (Takahashi et al. Proc. Natl. Acad. Sci. USA 1995, 92:1470-1474). These rats developed histological and serological changes comparable to HBV-induced acute hepatitis in humans. Intrahepatic injection of HBV was also performed in nude mice (Feitelson et al. J. Virol. 1988, 62:1408-1415). It was found that HBV infection in nude mice parallels that of HBV-infected chronic carriers (i.e., presence of virus antigens in blood and in the liver). These nude mice also develop lesions in the liver consistent with the presence of chronic hepatitis as seen in long term HBV infected patients. However, this model is not efficient for studying chronic HBV infection and assessing possible therapeutic treatments for chronic HBV infection due to large and uncontrolled variations in individual animals' viral gene expression, viremia, and liver disease.
Hepatitis C virus is also a leading cause of acute and chronic hepatitis, cirrhosis and hepatocellular carcinoma. HCV is a positive-strand RNA virus belonging to the Flaviviridae family, The genome of the virus comprises approximately 9500 nucleotides and contains a single open reading frame that spans the entire genome and encodes a large viral polypeptide of 3010 amino acids (Houghton et al. Hepatology 1991, 14:381-388). Upon infection of mammalian cells, RNA of the virus is translated into a single continuous polyprotein that is proteolytically processed by host signal peptidase and two viral proteases to produce at least 10 structural and non-structural proteins (Shimotohno et al. J. Hepatol. 1995, 22:87-92). The genome also contains a highly conserved 5' untranslated region that functions as an internal ribosome entry site for translation of the viral genome (Wang, C. and Siddiqui, A. Curr. Top. Microbiol. Immunol. 1995, 203:99-105). The HCV genome also contains a short 3' untranslated and a homopolymer tail of A or U residues after the open reading frame (Takamizawa et al. J. Virol. 1991, 65:1105-1113). A highly conserved 98 nucleotide non-homopolymeric sequence at the 3' end of the HCV RNA genome has also been identified (Kolykhalov et al. J. Virol. 1996, 70:3363-3371; Tanaka et al. J. Virol. 1996, 70:3307-3312).
An in vitro system for HCV propagation has been developed to characterize virus replication, virus persistence and viral pathogenicity. In this system, HCV replication was established in vitro by gene transfer of infectious HCV cDNA into HepG2 cells (Hiramatsu et al. J. Viral Hepatitis 1997, 4(Suppl. 1):61-67; Dash et al. American J. Pathology, 1997 151:363-373).
Further, several features of the human HCV infection have been found to be recapitulated in a chimpanzee model (Walker, C. M. Springer Semin. Immunopathol (Germany) 1997, 19(1):85-98). Frequency of persistent infection is high in both humans and chimpanzees. Viral replication also occurs despite evidence of cellular and humoral responses. However, the necroinflammatory lesions that develop in chronically infected chimpanzees are almost always mild, whereas in humans these lesions can range from mild to severe liver inflammation and endstage cirrhosis requiring transplantation. Further, the availability of this primate for research is strictly limited.
Accordingly, a number of transgenic mouse models for the study of HCV have been developed. For example, two independent transgenic mouse lines carrying the HCV core gene have been established to clarify whether or not the HCV core protein has an effect on pathological phenotypes in the liver (Moriya et al. J. Gen. Virol. 1997, 78(7):1527-31). These mice developed progressive hepatic steatosis, indicating that the HCV core protein plays a direct role in the development of hepatic steatosis, which characterizes hepatitis C.
Transgenic mice carrying the HCV gene envelope genes have also been shown to express the HCV envelope proteins in organs, including the liver and salivary glands (Koike et al. Proc. Natl Acad. Sci. USA 1997, 94(1):233-6). Further analysis of these animals has revealed that they develop exocrinopathy involving the salivary and lachrymal glands which resembles the pathology of Sjogren syndrome. Sjogren syndrome has been suggested to have a possible association with chronic hepatitis C. Accordingly, this transgenic mouse system is suggested to be a good animal model for the study of HCV infection.
However, there is a need for animal models that parallel the course of hepatitis virus infections in humans and approximate the evolution of a chronic carrier state which can be used in assessing possible therapeutic treatments for chronic infection by these viruses in humans.